A T-to-A substitution in the E1 alpha subunit gene of the branched-chain alpha-ketoacid dehydrogenase complex in two cell lines derived from Menonite maple syrup urine disease patients

We cloned and sequenced cDNAs of the E1 alpha and E1 beta subunits of the branched chain alpha-ketoacid dehydrogenase complex (BCKDH) in two cell lines derived from two different Menonite MSUD patients (GM 1655, GM 1099). A T-to-A substitution which generates an asparagine in place of a tyrosine at amino acid 394 of the mature E1 alpha subunit was present in both alleles in these two cell lines, whereas cDNAs of the E1 beta subunit in these cell lines were identical to that of normal human lymphoid cell line and that of the clone from a human placenta cDNA library. It is suggested that the Menonite MSUD is caused by the missense mutation of the E1 alpha subunit of the BCKDH complex.     

Nucleotide and deduced amino acid sequence of the E1 alpha subunit of human liver branched-chain alpha-ketoacid dehydrogenase

A 1552-bp cDNA for the E1 alpha subunit of branched-chain alpha-ketoacid dehydrogenase (BCKDH) was isolated from a human liver cDNA library. The cDNA contained a 1134-bp open reading frame that encoded 378 amino acid (aa) residues of the enzyme and 418 bp of 3'-untranslated sequence. The deduced amino acid sequence of the human protein shows 96% identity with that of the rat enzyme subunit. Those 117-aa residues surrounding the phosphorylation sites are completely conserved between man and rat. BCKDH E1 alpha showed considerable amino acid sequence similarity with pyruvate dehydrogenase E1 alpha, particularly in the region of the two principal phosphorylation sites of these proteins. Northern blots of human liver and skin fibroblasts demonstrated a single 1.8-kb mRNA band, with a higher level of E1 alpha mRNA in liver than in normal fibroblasts. Fibroblasts from a patient with thiamine-responsive maple syrup urine disease (MSUD) contained an mRNA of the same size and abundance as that of normal fibroblasts. Genomic DNA from normal and MSUD fibroblasts gave the same restriction maps on Southern blots, and the gene was approximately 10-kb in size.     

Isolation and characterization of a complementary DNA clone coding for the E1 beta subunit of the bovine branched-chain alpha-ketoacid dehydrogenase complex: complete amino acid sequence of the precursor protein and its proteolytic processing

A 1.7-kb cDNA clone encoding the entire precursor of the E1 beta subunit of the branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex was isolated from a bovine liver cDNA library by screening with a mixture of synthetic oligonucleotide probes corresponding to the C-terminal five-residue sequence of the mature E1 beta subunit. A partial amino acid sequence was determined by Edman degradation of the intact subunit and the peptides generated by cleavage at the lysyl bonds. Nucleotide sequence analysis revealed that the isolated cDNA clone contained the 5'-untranslated sequence of 186 nucleotides, the translated sequence of 1176 nucleotides, and the 3'-untranslated sequence of 306 nucleotides with a poly(A) tail. A type AATAAA polyadenylation signal was located 17 nucleotides upstream of the start of a poly(A) tail. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the partial amino acid sequence of the mature BCKDH E1 beta subunit showed that the cDNA insert encodes for a 342 amino acid subunit with Mr 37,745 and that the subunit is synthesized as the precursor with a leader sequence of 50 amino acids and processed at the N-terminus. Northern blot analysis using the cDNA insert as a probe showed the presence of a 1.8-1.9-kb mRNA in bovine liver, suggesting that the insert covers nearly a full length of mRNA. Alignment of the deduced amino acid sequence of bovine BCKDH E1 beta with that of the human pyruvate dehydrogenase (PDH) complex E1 beta subunit revealed a high degree of sequence homology throughout the two enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of mutations in Maple Syrup Urine Disease (MSUD): screening and identification of affected E1α and E1β subunits of the branched-chain α-keto-acid dehydrogenase multienzyme complex

Maple syrup urine disease (MSUD) is an autosomal recessive disease caused by a deficiency in subunits of the branched-chain α-keto-acid dehydrogenase complex (BCKDH). To characterize the mutations present in five patients with MSUD (four classic and one intermediate), three-step analyses were established: (1) identification of the involved subunit by complementation analysis using three different cell lines derived from homozygotes having E1α, E2β or the E2 mutant gene; (2), screening for a mutation site in cDNA of the corresponding subunit by RT-PCR-SSCP and (3), mutant analysis by sequencing the amplified cDNA fragment. Four single-base missense mutations, R115W, Q1556K, A209T and I282T, were detected in the E1α subunit. A single-base missense mutation H156R and three frame-shift mutations to generate stop codons downstream, including an 11-bp deletion of the tandem repeat in exon 1, a single-base (T) deletion and a single-base (G) insertion, were identified in the E1β subunit gene. All except one (11-bp deletion in E1β (Nobukini, Y., Mitsubuchi, H., Akaboshi, I., Indo, Y., Endo, F., Yoshioka, A. and Matsuda, I. (1991) J. Clin. Invest. 87, 1862–1866)) were novel mutations. The sites of amino-acid substitution were all conserved in other species. Thus, mutations causing MSUD are heterogeneous.